Multiple Conformations of Phosphodiesterase-5IMPLICATIONS FOR ENZYME FUNCTION AND DRUG DEVELOPMENT*

Received for publication, November 22, 2005, and in revised form, May 18, 2006 Published, JBC Papers in Press, May 30, 2006, DOI 10.1074/jbc.M512527200

Huanchen Wang‡, Yudong Liu‡, Qing Huai‡1, Jiwen Cai‡§, Roya Zoraghi¶, Sharron H. Francis¶, Jackie D. Corbin¶,Howard Robinson�, Zhongcheng Xin**, Guiting Lin‡‡, and Hengming Ke‡2

From the ‡Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina,Chapel Hill, North Carolina 27599-7260, §School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou,510080, China, the ¶Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine,Nashville, Tennessee 37232-0615, �Biology Department, Brookhaven National Laboratory, Upton, New York 11973-5000,**Andrology Center, Peking University First Hospital, Peking University, 8 Xishiku Street, Beijing (100034), China, and the‡‡Department of Urology, University of California, San Francisco, California 94143-1695

Phosphodiesterase-5 (PDE5) is the target for sildenafil, vard-enafil, and tadalafil, which are drugs for treatment of erectile dys-function and pulmonary hypertension.We report here the crystalstructures of a fully active catalytic domain of unligandedPDE5A1and its complexes with sildenafil or icarisid II. These structurestogether with the PDE5A1-isobutyl-1-methylxanthine complexshow that the H-loop (residues 660–683) at the active site ofPDE5A1 has four different conformations and migrates 7–35 Aupon inhibitor binding. In addition, the conformation of sildenafilreported herein differs significantly from those in the previousstructures of chimerically hybridized or almost inactive PDE5.Mutagenesis and kinetic analyses confirm that the H-loop is par-ticularly important for substrate recognition and that invariantGly659, which immediately precedes theH-loop, is critical for opti-mal substrate affinity and catalytic activity.

Cyclic nucleotide phosphodiesterases (PDEs)3 are keyenzymes controlling cellular concentrations of secondmessen-gers cAMP and cGMP by hydrolyzing them to 5�-AMP and5�-GMP, respectively. The human genome encodes 21 PDEgenes that are categorized into 11 families (1–9). AlternativemRNA splicing of the PDE genes produces over 60 PDE iso-forms that distribute in various cellular compartments and con-trol physiological processes. PDE molecules contain a con-served catalytic domain and variable regulatory regions.However, each PDE family possesses a characteristic pattern ofsubstrate specificity and inhibitor selectivity (6).Inhibitors of PDEs have been widely studied as therapeu-

tics as follows: cardiotonics, vasodilators, smooth musclerelaxants, antidepressants, antithrombotics, antiasthmatics,and agents for improving cognitive functions such as learn-ing andmemory (10–17). Some of the most successful exam-ples of these drugs are the PDE5 inhibitors sildenafil(Viagra�), vardenafil (Levitra�), and tadalafil (Cialis�) thathave been used for treatment of male erectile dysfunction (15).Sildenafil has also recently been approved (Revatio�) for treat-ment of pulmonary hypertension (18). However, reported sideeffects of these medications such as headache and visual dis-turbance suggest a need for further study of themolecular basisof the selectivity of PDE5 inhibitors (19).Two co-crystal structures of the catalytic domain of PDE5with

sildenafil showed differences in the conformation of the inhibitorbound to the catalytic site (20–22).However, it remains unknownwhether these conformations are biologically relevant because thePDE5 enzymeused in the studies is either almost inactive (20) or achimeric hybridof thePDE5catalytic domainwith replacementofa PDE4 segment (the H-loop) (21, 22). In addition, the crystalstructure of the catalytic domain of PDE5A1 in complex with thenonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX)showed that the conformation of the H-loop at the active site isdifferent from that in PDE4 (23) and in other published PDE5structures (20–22, 24). We report here the structure of the cata-lytic domain of human PDE5A1 in the unliganded state, as well asthe structures of the protein in complex with inhibitors sildenafiland icarisid II (Fig. 1). These structures, together with that ofPDE5A1-IBMX, reveal four different conformations of theH-loop, which is juxtaposed to the catalytic pocket of the enzyme.In addition, comparison of this PDE5-sildenafil structure with thepreviously published structures shows significantly different con-formations of the methylpiperazine portion of sildenafil. Theseunique features of the PDE5 catalytic domain and the sildenafilconfiguration are key considerations for understanding the actionof sildenafil and for development of PDE5 inhibitors.

EXPERIMENTAL PROCEDURES

Protein Expression and Purification of Catalytic DomainPDE5A1—The cDNA of the catalytic domain of humanPDE5A1 was generated by site-directed mutagenesis of thebovine PDE5A cDNA (23). The coding regions for aminoacids 535–860 of PDE5A1 were amplified by PCR and sub-

* This work was supported in part by National Institutes of Health GrantsGM59791 (to H. K.), DK58277, and DK40029 (to J. C.), and American HeartAssociation Postdoctoral Fellowship 032525B (to R. Z.). The costs of publi-cation of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked “advertisement” in accord-ance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 2H40, 2H42, and 2H44) havebeen deposited in the Protein Data Bank, Research Collaboratory for StructuralBioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

1 Present address: Center for Homeostasis and Thrombosis Research, BethIsrael Deaconess Medical Center and Dept. of Medicine, Harvard MedicalSchool, 41 Ave. Louis Pasteur, Boston, MA 02115.

2 To whom correspondence should be addressed. Tel.: 919-966-2244; Fax:919-966-2852; E-mail: hke@med.unc.edu.

3 The abbreviations used are: PDEs, phosphodiesterases; IBMX, 3-isobutyl-1-methylxanthine; r.m.s., root mean square.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 30, pp. 21469 –21479, July 28, 2006Printed in the U.S.A.

JULY 28, 2006 • VOLUME 281 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 21469

by guest on March 19, 2016

http://ww

w.jbc.org/

Dow

nloaded from

cloned into the expression vector pET15b. The resultantplasmid pET-PDE5A1 was transferred into Escherichia colistrain BL21 (Codonplus) for overexpression. The E. coli cellcarrying pET-PDE5A1 was grown in LB medium at 37 °C toan A600 � 0.7, and 0.1 mM isopropyl �-D-thiogalactopyrano-side was then added for further growth at 15 °C overnight.Recombinant PDE5A1 was purified by the columns of nick-el-nitrilotriacetic acid (Qiagen), Q-Sepharose, andSephacryl S300 (Amersham Biosciences). A typical purifica-tion yielded over 10 mg of PDE5A1 with a purity of �95%from a 2-liter cell culture.The mutant proteins with deletion of residues 663–678 and

661–681 were produced by the standard protocol of site-di-rected mutagenesis. For the deletion mutant proteins, four gly-cine residues were inserted as the spacer to minimize the dis-turbance on the three-dimensional structure. Overexpressionandpurification of themutant proteins used the sameprotocolsas for the nonmutated protein.Expression and Purification of Full-length hPDE5A1—Hu-

man cDNAcoding for full-length PDE5A1was cloned into pCR2.1-TOPO� vector (Invitrogen) and then ligated into the bacu-lovirus expression vector pAcHLT-A (Pharmingen). Theresulting plasmid pAcA-PDE5 (Met1–Asn875) was used tomake point mutations (G659A, V660Q, N661A, N662A,Y664A, H678A, and S679A) with theQuikChange site-directedmutagenesis kit (Stratagene). Wild type and mutant constructsof hPDE5A1 were expressed in Sf9 cells. Sf9 cells were cotrans-fected with BaculoGold linear baculovirus DNA (Pharmingen)and one of the pAcA-hPDE5A1 plasmids. The cotransfectionsupernatant was collected at 5 days post-infection, amplifiedthree times in Sf9 cells, and then used directly as virus stockwithout additional purification. Sf9 cells grown at 27 °C in com-plete Grace’s insect medium with 10% fetal bovine serum and10 �g/ml gentamicin (Sigma) were typically infected with 100�l of viral stock and then harvested at 92 h post-infection. Pro-tein yields were �2.7 mg/liter infected cell media and werelargely soluble.

The Sf9 cell pellet (�2 � 107 cells) was resuspended in ice-cold lysis buffer (20mMTris-HCl, pH 8, 100mMNaCl) contain-ing protease inhibitor mixture, homogenized, and centrifuged.The supernatant was loaded onto a nickel-nitrilotriacetic acid-agarose (Qiagen) column equilibrated with lysis buffer. Thecolumn was washed with lysis buffer containing a stepwise gra-dient of 0.8–20 mM imidazole and eluted with 100 mM imidaz-ole. Eluted PDE5A1 protein was dialyzed versus 2000 volumesof ice-cold 10 mM potassium phosphate, pH 6.8, 25 mM �-mer-captoethanol, and 150mMNaCl.All recombinant PDE5A1pro-teins exhibited a purity of �98% as determined by SDS-PAGE.Enzymatic Assay—Enzymatic activity of the isolated catalytic

domains of wild type PDE5A1 and its deletion mutants wasassayed by using [3H]cGMP as substrate in a reaction mixtureof 20 mM Tris-HCl, pH 7.8, 1.5 mM dithiothreitol, 10 mM

MgCl2, [3H]cGMP (40,000 cpm/assay) at 24 °C for 15 min (25).The reaction was terminated by addition of 0.2 M ZnSO4 andBa(OH)2. Radioactivity of unreacted [3H]cGMP in the superna-tant was measured by a liquid scintillation counter. The turn-over rate was measured at nine concentrations of cGMP andcontrolled at hydrolysis of 15–40% substrate. Each measure-ment was repeated three times. For measurement of IC50 val-ues, 10 concentrations of inhibitors were used at a substrateconcentration that was one-tenth of the Km value and anenzyme concentration that hydrolyzed 50% of substrate.To assay the activity of full-length hPDE5A1, the reaction

mixture contained 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.3mg/ml bovine serum albumin, 0.05–250 �M cGMP, and[3H]cGMP (100,000–150,000 cpm/assay) and one of thePDE5A1 proteins (26). Incubation time was 10 min at 30 °C. Inall studies, less than 10% of total [3H]cGMP was hydrolyzedduring the reaction. All values determined represent threemeasurements. The Km and kcat values for cGMP were deter-mined by nonlinear regression analysis of data using PrismGraphPad software.Crystallization and Data Collection—All crystals of

PDE5A1-(535–860) were grown by vapor diffusion. The pro-

FIGURE 1. Chemical structures of PDE5 inhibitors. The letters R1–R3 label the main groups in sildenafil and icarisid II. In sildenafil, R1 represents the pyrazo-lopyrimidinone group; R2 is ethoxyphenyl, and R3 is methylpiperazine. In icarisid II, R1 represents the methoxyphenyl group; R2 is oxychromone, and R3 isrhamnose.

Structures of Phosphodiesterase-5

21470 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 30 • JULY 28, 2006

by guest on March 19, 2016

http://ww

w.jbc.org/

Dow

nloaded from

tein drop was prepared by mixing 2 �l of protein with 2 �l ofwell buffer. The unliganded PDE5A1 crystal was grown at roomtemperature against well buffer of 0.2 MMgSO4, 0.1 MTris base,pH 8.5, 12% PEG 3350, and 2% ethanol. The PDE5A1-sildenafilcomplexwas prepared bymixing 1mM sildenafil with 15mg/mlPDE5A1 at 4 °C overnight and crystallized against a well bufferof 1.0 M sodium citrate, 2.5% ethanol, 0.1 M HEPES, pH 7.5, at4 °C. The PDE5A1-icarisid II complex was prepared by mixing2 mM icarisid II with 15 mg/ml protein at 4 °C overnight, andcrystallized against a well buffer of 0.1 M HEPES, pH 7.5, 12%PEG3350 at room temperature.The unliganded PDE5A1-(535–860) was crystallized in the

space group P3121with cell dimensions of a� b� 74.7 and c�130.7 Å. The PDE5A1-sildenafil crystal had the space groupP6222 with cell dimensions of a � b � 164.6 and c � 193.1 Å.The PDE5A1-icarisid II crystal had the space group P6122 withcell dimensions of a� b� 110.7 and c� 106.2Å. BeamlineX25at Brookhaven National Laboratory was used for collection ofdiffraction data of the unliganded PDE5A1 and X29 forPDE5A1-sildenafil and PDE5A1-icarisid II (Table 1). All datawere processed by the program HKL (27).Structure Determination—The structure of the unligan-

ded PDE5A1 was solved by rigid body refinement of thePDE5A1 catalytic domain in PDE5A1-IBMX. The structuresof PDE5A1 in complex with sildenafil and icarisid II weresolved by the molecular replacement program AMoRe (28),using the PDE5A1-IBMX structure without the H-loop andIBMX as the initial model. The rotation and translationsearches for the crystal of PDE5A1-icarisid II yielded a correla-tion coefficient of 0.74 and R-factor of 0.31 for 3054 reflectionsbetween 4 and 8 Å resolution. The rotation and translationsearches for PDE5A1-sildenafil yielded a correlation coefficientof 0.22 andR-factor of 0.52 for 11,612 reflections between 4 and8 Å resolution for the first molecule, and of 0.39 and 0.41 afterthe second molecule was added. The electron density map was

improved by the density modification package of CCP4 (29).The atomicmodel was rebuilt by programO (30) and refined byprogram CNS (Table 1) (31).

RESULTS

Multiple Conformations of the H-loop of PDE5—The crystal-lographic asymmetric units contain one molecule of the cata-lytic domain in the structures of the unliganded PDE5A1 andthe icarisid II complex but three molecules in PDE5A1-silde-nafil structure. The electron density maps showed that theentire catalytic domain in the PDE5A1-icarisid II structure andmolecule A in the PDE5A1-sildenafil structure were traceable.However, residues 668–676 of molecules B and C in thePDE5A1-sildenafil crystal and residues 793–807 in the unli-ganded PDE5A1 lacked electron density and were disordered.The Ramachandran plots showed that the backbone conforma-tions of 90–94% residues in the three structures were located inthe most favored regions, and no residues were located in theenergetically disallowed regions.The structures of the unliganded PDE5A1 catalytic domain

(residues 535–860) and its complexes with sildenafil or icarisidII are composed of 14 common �-helices and a variable H-loopat the active site (Fig. 2). Structural superposition of the unli-ganded PDE5A1 over the complexes of PDE5A1-IBMX,PDE5A1-sildenafil, and PDE5A1-icarisid II yielded r.m.s. devi-ations of 0.29, 0.42, and 0.54 Å for the C� atoms of comparableresidues (536–659, 684–787, and 810–860), respectively, sug-gesting overall structural similarity. However, the H-loop (res-idues 660–683 on the basis of the structural comparison withseven other PDE families, which are slightly different from theprevious assignment of residues 661–676 (23)) adopts fourconformations anddifferent tertiary structures in the crystals ofthe unliganded PDE5A1 and its complexes with IBMX, silde-nafil, or icarisid II. In the unliganded PDE5A1 structure, theH-loop contains a few turns, but the majority of the H-loop

TABLE 1Statistics on diffraction data and structure refinement

Data collection PDE5A1 native PDE5A1-sildenafil PDE5A1-icarisid IISpace group P3121 P6222 P6122Unit cell (a, b, and c, Å) 74.7, 74.7, 130.7 164.6, 164.6, 193.1 110.7, 110.7, 106.2Resolution (Å) 1.85 2.3 1.8Total measurements 363,960 1,121,219 412,999Unique reflections 36,603 68,786 36,177Completeness (%) 99.5 (100.0)a 99.9 (100.0) 99.9 (99.7)Average I/� 20.8 (7.4)a 6.7 (4.8) 13.6 (4.3)Rmerge 0.058 (0.49)a 0.117 (0.58) 0.061 (0.46)Structure refinementR-factor 0.221 0.210 0.206R-free 0.236 (9.7%)b 0.246 (9.7%) 0.233 (9.7%)Resolution (Å) 15–1.85 48–2.3 44–1.8Reflections 34,996 66,315 35,094

r.m.s. deviationBond 0.0078 Å 0.0061 0.0073Angle 1.25° 1.17° 1.31°

Average B-factor (Å2)Protein 37.5 (2523)c 27.8 (7804) 27.9 (2650)Inhibitor 29.4 (99) 25.9 (37)Water 37.9 (148)c 28.8 (311) 32.8 (231)Zinc 46.2 (1)c 44.9 (3) 36.6 (1)Magnesium 33.4 (1)c 37.6 (3) 41.0 (1)

a The numbers in parentheses are for the highest resolution shell.b The percentage of reflections omitted for calculation of R-free.c The number of atoms in the crystallographic asymmetric unit.

Structures of Phosphodiesterase-5

JULY 28, 2006 • VOLUME 281 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 21471

by guest on March 19, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Structures of Phosphodiesterase-5

21472 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 30 • JULY 28, 2006

by guest on March 19, 2016

http://ww

w.jbc.org/

Dow

nloaded from

residues are in a coil conformation (Fig. 2). Binding of IBMXconverts the H-loop into two short �-helices involving residues664–667 and 672–676 (Fig. 2F) and shifts the C� atoms of theH-loop as much as 7 Å from those in the unliganded structure.In the structure of the PDE5-sildenafil complex, the H-loop inmolecule A is converted to a turn and a 310 helix at residues672–675, and the whole loopmigrates asmuch as 24Å to coverthe active site (Fig. 2). However, residues 668–676 of theH-loop in molecules B and C are disordered. The most dra-matic change in the H-loop occurs in the structure of PDE5A1-icarisid II, in which the H-loop contains two �-strands at resi-dues 662–666 and 675–679 (Fig. 2F) and migrates as much as35 Å from the position in the unliganded PDE5A1.To verify that the conformational changes are not because of

an artifact of structure determination or crystal packing, elec-tron density maps were calculated, and lattice interactions inthe various crystal forms were examined. The maps that werecalculated from the structure with omission of the H-loopshowed solid electron density for almost all residues of theH-loops, thus confirming the true conformational variation inthe PDE5A1 structures. This is supported by the fact that theB-factor for the H-loops is comparable with or slightly higherthan the overall average B-factor for the protein atoms as fol-lows: 48 versus 38 Å2 for the unliganded PDE5A1, 61 versus 40Å2 for PDE5A1-IBMX, 33 versus 33 Å2 for PDE5A1-icarisid II,and 38 versus 27 Å2 for PDE5A1-sildenafil. In addition, the fol-lowing facts suggest minor roles of the lattice contacts in theconformational changes of the H-loop. First, the unligandedPDE5A1 and its IBMX complex have the same space group andthe similar unit cell parameters (a � b � 74.7, c � 130.7 Åversus a � b � 74.5, c � 130.1 Å) but different H-loop confor-mations. Second, the PDE4 H-loop that was inserted into thechimeric PDE5 structure is involved in the crystal lattice inter-actions of PDE5 but retains its conformation found in PDE4(22). Thus, the dramatic conformational changes of the H-loopmust be the consequence of binding of the specific inhibitors.In addition to variation of the H-loop, minor conformational

differences are observed for another active site loop, theM-loop (residues 788–811 on the basis of the structural com-parison among seven PDE families, in comparison to the orig-inal assignment of residues 787–812 (23)). Residues 793–807of theM-loop are not traceable in the structures of the unligan-ded or IBMX-bound PDE5A1. However, the well orderedM-loops in the structures of PDE5A1 in complexwith sildenafilor icarisid II contain an extra 310 helix and a 10-residue exten-sion of �-helix H14, in addition to the correspondence of a 310helix to the N-terminal portion of H15 in the unligandedPDE5A1 (Fig. 2F).PDE5 shows an apparently unique feature distinct from

other PDE families, although the overall topological folding ofPDE5A is similar to those of PDE1B (22), PDE2A (32), PDE3B

(33), PDE4B and PDE4D (22–24, 34–38), PDE7A (25), andPDE9A (39). The core catalytic domains of PDE1–4, -7, and -9(residues 115–411 in PDE4D2), including the H-loop that iscomposed of two short�-helices (H8 andH9, residues 209–215and 218–222 in PDE4D2l; see Fig. 2F) have a uniform confor-mation and are superimposable on one another. In contrast, theH-loop of PDE5A1 presents at least four different conforma-tions, and none of these is comparable with any of the corre-sponding H-loops in other PDE families. The closest compara-ble conformation is the H-loop in the PDE5A1-IBMXstructure, which also contains two short �-helices. However,these twohelices have asmuch as 7Åpositional difference fromthose in PDE4D2 (23) and are in a different three-dimensionalarrangement. In addition, the �-strand component of theH-loop in the PDE5A1-icarisid II complex is unique amongactive sites of known PDE structures. Therefore, the active siteof PDE5 appears to belong to a special category of the PDEsuperfamily.Conformation Variation of Sildenafil—Sildenafil binds to each

active site of three PDE5A1 catalytic domains in the crystallo-graphic asymmetric unit with similar conformation and occu-pancy, as shownby thecomparableB-factor and thecleanelectrondensity in theomittedmap (Fig. 3).Thebindingof sildenafil causesa dramatic conformational change of the H-loop and a move-ment as much as 24 Å from that in the unliganded PDE5 struc-ture. A direct consequence of the H-loop movement is thetransformation of the open PDE5A1 active site to a closedpocket. Sildenafil is partially buried in the pocket. Solvent-ac-cessible surface of sildenafil after binding to PDE5A1 is reducedto 9.4% of the total surface area. Sildenafil borders the metal-binding pocket but does not directly interact with the metalions. The pyrazolopyrimidinone group (R1 in Fig. 1 andTable 2)of sildenafil stacks against Phe820 of PDE5A1 and also contactsresidues Tyr612, Leu765, Ala767, and Gln817. The O1 and N4atoms of pyrazolopyrimidinone form two hydrogen bonds withN�2 and O�1 of Gln817, respectively. The ethoxyphenyl group(R2, Fig. 1) interacts via van derWaals forceswithVal782, Ala783,Phe786, Leu804, Ile813, Gln817, andPhe820. Themethylpiperazinegroup (R3, Fig. 1) contacts Asn662, Ser663, Tyr664, Ile665, Leu804,and Phe820. The oxygen atoms of the sulfone group interactmainly with Phe820.Our PDE5A1-sildenafil structure is similar in many respects

to those reported earlier (20–22), as shown by the r.m.s. devia-tions of 0.59 and 0.40 Å for superposition of the backboneatoms of the comparable residues (without 661–677) of ourPDE5A1-sildenafil over the two previous structures. However,two significant differences are observed among the threePDE5A-sildenafil structures. First, the H-loop in our structurehas definite electron density and different conformation fromthose in the previously published structures in which theH-loop is either disordered (missing residues 665–675) (20) or

FIGURE 2. Structures of PDE5 and its inhibitor complexes. A, ribbon diagram. The cyan ribbons are the common structures of the catalytic domains ofunliganded PDE5A1 and its complexes. The different conformations of the H-loop in the four structures are shown in purple for the unliganded PDE5A1,blue for the IBMX complex, gold for the sildenafil complex, and green for the icarisid II complex. Zinc is colored red, and magnesium is purple. B–E, surfacepresentation for the catalytic domain of the unliganded PDE5A1 (B) and its complexes with IBMX (C), sildenafil (D), or icarisid II (E). The middle red pocketis the active site. The inhibitors are shown as color balls: blue for IBMX, gold for sildenafil, and green for icarisid II. Icarisid II is almost completely buried,and the orientation of the domain is adjusted to show the icarisid II binding. F, the alignment of the secondary structures with the amino acid sequencearound the regions of the H- and M-loops that are shown in blue letters. The gold ribbon, green arrow, and cyan tube represent 310 helix, �-strand, and�-helix, respectively. The dots represent disordered regions in the unliganded and IBMX-bound PDE5.

Structures of Phosphodiesterase-5

JULY 28, 2006 • VOLUME 281 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 21473

by guest on March 19, 2016

http://ww

w.jbc.org/

Dow

nloaded from

FIGURE 3. Sildenafil binding. A, electron density for sildenafil bound to PDE5A1. The Fo � Fc map was calculated from the structure omitting sildenafil andcontoured at 3�. B, stereoview of interactions of sildenafil (gold stick-balls) with the active site residues (salmon sticks). The dotted lines represent hydrogenbonds. C, superposition of sildenafils from the PDE5A1-sildenafil structures of Zhang et al. (22) (gold), Sung et al. (20) (cyan), and ours (green).

Structures of Phosphodiesterase-5

21474 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 30 • JULY 28, 2006

by guest on March 19, 2016

http://ww

w.jbc.org/

Dow

nloaded from

takes the PDE4 conformation because of chimeric replacementof PDE5 residues 658–681with those of PDE4 (22). Second, theconformations and the interactions of sildenafil in the threePDE5A-sildenafil structures are significantly different.Although the ethoxyphenyl and pyrazolopyrimidinone groupsof sildenafil in the three PDE5A structures are superimposableand interact with the same residues of PDE5A, the meth-ylpiperazine shows different orientations (Fig. 3). The meth-ylpiperazinemoiety in our PDE5A1-sildenafil structure folds tointeract with the pyrazolopyrimidinone, in contrast to the con-formation reported by Sung et al. (20) where the methylpipera-zine extends in a different direction. The orientation of themethylpiperazine in the structure of Zhang et al. (22) is moresimilar to ours, but the ring is tilted by�40° (Fig. 3). In addition,the contacts between the methylpiperazine and the PDE5 resi-dues in the structure reported hereinmarkedly differ frombothpreviously published structures. In the structure reportedherein, the methylpiperazine interacts with Asn662, Ser663,Tyr664, and Ile665 in theH-loop (Table 2).However, there are nocontacts between the methylpiperazine and the H-loop in thestructure of Zhang et al. (22), and the methylpiperazine con-tacts Tyr664,Met816, Gly819, and Phe820 in the structure by Sunget al. (20).To exclude possible false results from the structure determi-

nation, we compared this study and previously published struc-tures of the PDE5A-sildenafil complex. The 2Fo � Fc and Fo �Fc maps that are calculated from the PDE5A catalytic domainswith omission of sildenafil show reasonable electron density forall three sildenafils, suggesting their true bound conformationsin the crystal states. Thus, the basis for the conformational dif-ferences of sildenafil needs to be explored. The 40° tilt of themethylpiperazine ring in the Zhang structure may be the con-sequence of the chimeric replacement of the PDE5H-loop (res-idues 658–681) with the equivalent amino acids of PDE4 (22),which takes the conformation of PDE4 in that structure. For thestructure of PDE5A-sildenafil of Sung et al. (20), the electron

density maps show that Cys677 probably forms an intermolec-ular disulfide bondwithCys677 fromaneighboringmonomer inthe PDE5A crystal. Thus, the catalytic domain of PDE5A in thestructure of Sung et al. (20) forms a dimer, in contrast to amonomeric form in the PDE5A-sildenafil structures of oursand Zhang et al. (22). The fact that the specific activity of thecatalytic domain of Sung et al. (20) is only about one-thou-sandth of that reported for monomeric PDE5A (see Ref. 40 andthis study) suggests that the dimer may be either a less activeform of PDE5 or a crystallization artifact.Binding of Icarisid II to PDE5—Icarisid II is a glycoside deriv-

ative of flavonoids from the plantEpimediumwanshanense thathas been used as an herbal medicine for improvement of erec-tile dysfunction in China for more than a thousand years (41–44). Icarisid II inhibits PDE5A1 with an IC50 of 2 �M and showsat least 10-fold selectivity against other PDEs. It binds to theactive site of PDE5A1, as shown by the electron density that iscalculated from the PDE5A1 structure before icarisid II wasbuilt into the structure (Fig. 4). The binding of icarisid II causesformation of two �-strands in the H-loop and as much as 35 Åmovement of the H-loop to completely close the active site(Figs. 2E and 4). The solvent-accessible area of icarisid II afterbinding to the active site of PDE5A1 is reduced to 0.05% of theunbound form.Five hydrogen bonds are formed between icarisid II and

PDE5A1. The oxychromone (R2, Fig. 1 andTable 2) of icarisid IIstacks against Phe820, and its oxygen atoms O4 and O10 formthree hydrogen bonds with the backbone nitrogen of Ile665, theside chain oxygen of Ser668, and a water molecule. It also inter-acts with residues Tyr664, Leu725, Leu804, and Met816 (Fig. 4).The methoxyphenyl group (R1, Fig. 1) forms a hydrogen bondwith the backbone nitrogen of Ile768 and makes van der Waalscontacts with Ala767, Ile768, Gln775, Ala779, Gln817, and Phe820(Table 2). The pentenyl group (atoms C17-C21, Fig. 1) formshydrophobic contacts with Val782, Phe786, Leu804, Met816, andGln817. The rhamnose group (R3, Fig. 1) forms three hydrogen

TABLE 2Interactions of sildenafil and icarisid II with PDE5A

Structures of Phosphodiesterase-5

JULY 28, 2006 • VOLUME 281 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 21475

by guest on March 19, 2016

http://ww

w.jbc.org/

Dow

nloaded from

FIGURE 4. Icarisid II binding. A, electron density for icarisid II bound to PDE5A1. The Fo � Fc map was calculated from the structure with omission of icarisid IIand contoured at 3.0�. B, stereoview of interaction of icarisid II (gold sticks) with the PDE5A1 residues (salmon sticks) at the active site of PDE5A1. The dotted linesrepresent hydrogen bonds. C, superposition of icarisid II (gold sticks) and sildenafil (purple ball-sticks). Residues from PDE5A-sildenafil are shown in blue, andresidues from the icarisid II complex are in green. The side chain of Gln817 has the different conformations in the two structures.

Structures of Phosphodiesterase-5

21476 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 30 • JULY 28, 2006

by guest on March 19, 2016

http://ww

w.jbc.org/

Dow

nloaded from

bonds with N�2 of His613, O�2 of Asp764, and a water molecule,in addition to interactions with residues Tyr612, His613, Asn661,Ser663, Leu725, Asp764, Leu765, and Phe820 (Table 2). A fewatoms of rhamnose are located at distance suitable for van derWaals contact with both metal ions and directly interact withsome of the metal-binding residues such as Asp764.Although icarisid II and sildenafil occupy the same active

site, the detailed interactions of the inhibitors are significantlydifferent (Fig. 4). Sildenafil forms two hydrogen bonds withGln817 and stacks against Phe820. However, Gln817 shows about90° rotation of its side chain (Fig. 4) and forms no hydrogenbond with icarisid II. The hydrogen bond between the sidechain atoms of O�1 of Gln775 and N�2 of Gln817 in the unligan-ded PDE5 and its complex with IBMX and sildenafil vanishes inthe PDE5-icarisid II structure, and the two atoms separate by4.3 Å. The conformational change of the side chain Gln817 inthe icarisid II structure appears to be a consequence of its unfa-vorable proximity to the hydrophobic pentenyl and phenylgroups. In addition, the position of oxychromone of icarisid II,which stacks against Phe820, is significantly different from thepyrazolopyrimidinone of sildenafil. In consideration of the1000-fold difference in binding affinity between sildenafil andicarisid II, the structural data suggest that the hydrogen bondwith Gln817 and the stacking against Phe820 are two essentialcomponents for high affinity binding of inhibitors. This result isconsistent with an established role of Gln817 in providing forhigh potency of sildenafil, vardenfil, and tadalafil (45).

A Potential Role of the H-loop in Substrate/InhibitorRecognition—To understand the biochemical basis of multipleconformations of the H-loop, two deletion mutants were cre-ated in the isolated catalytic domain of PDE5A1. The PDE5A1mutant with deletion of residues 663–678 and insertion of fourglycines (to minimize perturbation of the structure) showedabout 10- and 2-fold lower affinity for cGMP and the inhibitors,respectively (Table 3 and Fig. 5). The mutant protein in whichresidues 661–681 had been deleted and four glycines had beeninserted had a 150-fold weakerKm value for cGMP and 30–80-fold less potent IC50 value for the inhibitors (Table 3). However,both mutants had kcat values comparable with that of the wildtype PDE5A1 catalytic domain. These data imply that theH-loop has a very important role for interaction of PDE5A1with the cGMP substrate but appears to be less critical in inter-action with icarisid II or sildenafil than with cGMP. This isconsistent with an earlier report that a sildenafil homologUK-122764 that lacks methylpiperazine and thus interac-tions with the H-loop shows only a 5-fold lower potency thansildenafil (46).To pinpoint residues that impact cGMP binding affinity, sin-

gle mutations were performed on selected H-loop residues(Val660, Asn661, Asn662, Tyr664, His678, and Ser679) in full-lengthhuman PDE5A1, in addition to Gly659, an invariant amino acidamong all class I PDEs. Mutation of Gly659 to alanine (G659A)caused a 17-fold loss of kcat and 24-fold weaker affinity forcGMP (Table 4). The N662A mutant exhibited a modest loss(5-fold) in affinity for cGMP and a 9-fold decrease in kcat.Muta-tion ofH678Adid not appreciably impact the binding affinity ofsubstrate or catalytic activity. The remaining mutations(V660Q, N661A, Y664A, and S679A) did not significantlychange kcat but caused a 24–28-fold loss in affinity for cGMP(Table 4). To verify the overall structural integrity of the full-length PDE5A1 constructs, cGMP binding to the allostericcGMP sites in the regulatory domain of each mutant wasassessed.Wild type andmutant PDE5A1proteins bound cGMPwith comparable affinity (Kd in the range of 190–230 nM) andstoichiometry (0.50–0.55 mol of cGMP per PDE5A1 mono-mer), in good agreement with values reported previously for

FIGURE 5. Inhibition of the isolated PDE5 catalytic domain of wild type and H-loop deletion mutants by IBMX, icarisid II, and sildenafil.

TABLE 3Kinetic properties of PDE5A1-(535– 860) isolated catalytic domain and its mutants

KmcGMP kcatcGMP (kcat/Km)cGMP IBMX Icarisid II Sildenafil�M s�1 s�1 �M�1 IC50 �M IC50 �M IC50 nM

Wild type 5.1 � 1.3 1.3 � 0.3 0.27 � 0.08 2.1 � 0.5 1.7 � 0.4 2.4 � 0.3�(663–678)a 54 � 6 3.8 � 0.5 0.07 � 0.013 5.3 � 1.1 2.5 � 0.2 5.9 � 1.5�(661–681)a 750 � 210 2.3 � 0.6 0.0031 � 0.0002 95 � 6 55 � 13 188 � 29

a Four glycines were inserted as a linker. Each of the experiments was repeated three times.

TABLE 4Kinetic properties of full-length wild type PDE5A1 and PDE5A1mutant proteins

KmcGMP (kcat/Km)cGMP (kcat/Km)cGMP

�M s�1 s�1 �M�1

Wild type 2.95 � 0.81 1.67 � 0.31 0.57G659A 71.20 � 1.22 0.112 � 0.00013 0.0016V660Q 82.06 � 8.46 2.53 � 0.71 0.031N661A 68.06 � 4.32 0.86 � 0.04 0.013N662A 15.15 � 1.92 0.18 � 0.10 0.012Y664A 80.36 � 8.76 1.51 � 0.35 0.019H678A 3.87 � 0.10 0.53 � 0.06 0.14S679A 70.28 � 5.45 1.83 � 0.37 0.026

Structures of Phosphodiesterase-5

JULY 28, 2006 • VOLUME 281 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 21477

by guest on March 19, 2016

http://ww

w.jbc.org/

Dow

nloaded from

recombinant bovine PDE5 (40). These data indicate that overallstructures of the mutant proteins are preserved and that differ-ences in kinetic parameters for the mutants are not because ofnonspecific conformational effects.Properties of the G659Amutant can be explained on basis of

the PDE structures. Gly659 is invariant in all class I PDE families,immediately precedes the H-loop, and is likely to provide crit-ical flexibility at this juncture in the protein structures. Gly659has backbone conformational angles of � � 76–105°, � �3–22°, and � �180° in the structures of PDE5A1-sildenafil,PDE5A1-icarisid II, and other PDE families, and� � 104–109°,� � 139–141°, and � �180° in the unliganded and IBMX-bound PDE5A1. All of these �/� conformational angles areallowed only for glycine but are not allowed for all other aminoacids. Thus, mutation of Gly659 to any other amino acid wouldimpose restrictions on the conformation of the H-loop or evendistort the conformation of the active site, thus profoundlyimpairing the catalytic activity of the mutant PDE5.The kinetic data indicate that the majority of the H-loop

mutants affect Km values for cGMP but have much less effectin catalytic turnover rate (Table 4). Although no interactionsbetween the H-loop and the low affinity product GMP wereobserved in the chimeric PDE5-GMP structure (22), it isplausible that the H-loopmay contact cGMP in a pattern likethat of sildenafil or icarisid II. Because hydrolysis of thephosphodiester bond of cGMP occurs in the metal-bindingsubpocket, the distal position of the H-loop from the metalsite in the structure would be more consistent with its role insubstrate binding rather than hydrolysis. This would imply adynamic interplay between the H-loop and substrate, inwhich binding of cGMP and the H-loop conformation wouldmutually regulate one another. Further structural studieswill be required to completely define the mechanisminvolved in the H-loop modulation of substrate affinity andinhibitor binding.

DISCUSSION

Extensive studies on the crystal structures of PDEs haveshown that the PDE families have similar overall three-dimen-sional structures for their isolated catalytic domains and activesites (20–25, 32–39). However, information on the conforma-tion of the PDE5 active site and on sildenafil binding is incom-plete because early studies showed a disordered or artificiallyreplaced H-loop at the active site (20–22). This study, in com-bination with our PDE5A1-IBMX structure (23), reveals thatthe H-loop of PDE5 can adopt four clearly defined conforma-tions. These different conformations of the H-loop of PDE5may be the result of direct contacts between the H-loop andinhibitors, such as those in the structures of PDE5A1 in com-plex with sildenafil or icarisid II. Alternatively, the H-loopchanges may be imposed by more distant effects of conforma-tional changes following inhibitor occupation of the bindingpocket, as implicated by no direct contacts between the H-loopand IBMX. Finally, a combination of both direct and indirectinteractions may contribute to the H-loop changes. Because allother PDEs appear to have a similar conformation of theH-loopthat is not comparable with any of the conformations of thePDE5 H-loop, the PDE5 active site apparently has a unique

structural characteristic. The mutual communication betweeninhibitor binding and conformational changes of the PDE5 cat-alytic pocket may thus be a valuable consideration for design ofnew inhibitors with unique selectivity against PDE5.It is common that an inhibitor slightly adjusts its conforma-

tion to provide an optimal fit in the binding pocket of a protein.However, the conformational variation of sildenafil in the dif-ferent crystal forms, as seen in the significantly different con-formations of the methylpiperazine group, is unusual. Becauseinhibitors are commonly designed tomimic contacts employedby the substrate, the dramatic effect of the H-loop mutationson affinity for cGMP and the flexibility of sildenafil providepotentially important direction in development of new PDE5inhibitors.Side effects such as visual disturbances in patients who ingest

PDE5 inhibitors (19) dictate a need for detailed study of themolecular basis for the action of these drugs and developmentof new potential inhibitors. Derivatives of flavonoids such asicarasid II may be such a new category of PDE5 inhibitors. Fla-vonoids inhibit PDEs with affinity at the micromolar level andslight selectivity (47, 48), and are widely used as dietary supple-ments that have reached a multiple billion dollar business (49–51). In the present report, the structure of PDE5A1-icarisid IIshows how this natural dietary compound interacts with PDE5and thus provides a valuable guideline for development of a newcategory of PDE5 inhibitors.

Acknowledgment—We thank the National Synchrotron Light Sourcefor collection of diffraction data. We especially thank Drs. KenjiOmori and Jun Kotera of Tanabe-Seiyaku Pharmaceutical Co. Ltd.(Saitama, Japan) for kindly providing human PDE5A1 cDNA. Wealso thank Meiyan Zheng and Kennard Grimes for their excellenttechnical assistance.

REFERENCES1. Manganiello, V. C., Taira, M., Degerman, F., and Belfrage, P. (1995) Cell.

Signal. 7, 445–4552. Houslay, M. D., Sullivan, M., and Bolger, G. B. (1998)Adv. Pharmacol. 44,

225–3433. Torphy, T. J. (1998) Am. J. Respir. Crit. Care Med. 157, 351–3704. Soderling, S. H., and Beavo, J. A. (2000)Curr. Opin. Cell Biol. 12, 174–1795. Francis, S. H., Turko, I. V., and Corbin, J. D. (2001) Prog. Nucleic Acids Res.

Mol. Biol. 65, 1–526. Mehats, C., Andersen, C. B., Filopanti, M., Jin, S. L., and Conti, M. (2002)

Trends Endocrinol. Metab. 13, 29–357. Conti, M., Richter,W., Mehats, C., Livera, G., Park, J. Y., and Jin, C. (2003)

J. Biol. Chem. 278, 5493–54968. Houslay, M. D., and Adams, D. R. (2003) Biochem. J. 370, 1–189. Goraya, T. A., and Cooper, D. M. (2005) Cell. Signal. 17, 789–79710. Movsesian, M. A. (2000) Exp. Opin. Investig. Drugs 9, 963–97311. Truss,M. C., Stief, C. G., Uckert, S., Becker, A. J.,Wafer, J., Schultheiss, D.,

and Jonas, U. (2001)World J. Urol. 19, 344–35012. Liu, Y., Shakur, Y., Yoshitake, M., and Kambayashi, J. J. (2001)Cardiovasc.

Drug Rev. 19, 369–38613. Huang, Z., Ducharme, Y., MacDonald, D., and Robinchaud, A. (2001)

Curr. Opin. Chem. Biol. 5, 432–43814. Rotella, D. P. (2002) Nat. Rev. Drug Discovery 1, 674–68215. Corbin, J. D., and Francis, S. H. (2002) Int. J. Clin. Pract. 56, 453–45916. Lipworth, B. J. (2005) Lancet 365, 167–17517. Castro, A., Jerez, M. J., Gil, C., and Martinez, A. (2005)Med. Res. Rev. 25,

229–244

Structures of Phosphodiesterase-5

21478 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 30 • JULY 28, 2006

by guest on March 19, 2016

http://ww

w.jbc.org/

Dow

nloaded from

18. Galie, N., Ghofrani, H. A., Torbicki, A., Barst, R. J., Rubin, L. J., Badesch, D.,Fleming, T., Parpia, T., Burgess, G., Branzi, A., Grimminger, F., Kurzyna,M., and Simonneau, G. (2005) N. Engl. J. Med. 353, 2148–2157

19. Pomeranz, H. D., and Bhavsar, A. R. (2005) J. Neuroophthalmol. 25, 9–1320. Sung, B. J., Hwang, K. Y., Jeon, Y. H., Lee, J. I., Heo, Y. S., Kim, J. H., Moon,

J., Yoon, J. M., Hyun, Y. L., Kim, E., Eum, S. J., Park, S. Y., Lee, J. O., Lee,T. G., Ro, S., and Cho, J. M. (2003) Nature 425, 98–102

21. Brown, D. G., Groom, C. R., Hopkins, A. L., Jenkins, T. M., Kamp, S. H.,O’Gara, M.M., Ringrose, H. J., Robinson, C.M., and Taylor,W. E. (May 8,2003) WIPO Patent WO 03/2003/038080

22. Zhang, K. Y., Card, G. L., Suzuki, Y., Artis, D. R., Fong, D., Gillette, S.,Hsieh, D., Neiman, J., West, B. L., Zhang, C., Milburn, M. V., Kim, S. H.,Schlessinger, J., and Bollag, G. (2004)Mol. Cell 15, 279–2861

23. Huai, Q., Liu, Y., Francis, S. H., Corbin, J. D., and Ke, H. (2004) J. Biol.Chem. 279, 13095–13101

24. Card, G. L., England, B. P., Suzuki, Y., Fong, D., Powell, B., Lee, B., Luu, C.,Tabrizizad,M., Gillette, S., Ibrahim, P. N., Artis, D. R., Bollag, G.,Milburn,M. V., Kim, S. H., Schlessinger, J., and Zhang, K. Y. (2004) Structure(Camb.) 12, 2233–2247

25. Wang, H., Liu, Y., Chen, Y., Robinson, H., and Ke, H. (2005) J. Biol. Chem.280, 30949–30955

26. Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) J. Biol. Chem. 265,14971–14978

27. Otwinowski, Z., and Minor, W. (1997)Methods Enzymol. 276, 307–32628. Navaza, J., and Saludjian, P. (1997)Methods Enzymol. 276, 581–59429. Collaborative Computational Project, Number 4 (1994) Acta Crystallogr.

Sect. D Biol. Crystallogr. 50, 760–76330. Jones, T. A., Zou, J.-Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta

Crystallogr. Sect. A 47, 110–11931. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P.,

Grosse-Kunstleve, R.W., Jiang, J. S., Kuszewski, J., Nilges,M., Pannu,N. S.,Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) ActaCrystallogr. Sect. D Biol. Crystallogr. 54, 905–921

32. Iffland, A., Kohls, D., Low, S., Luan, J., Zhang, Y., Kothe, M., Cao, Q.,Kamath, A. V., Ding, Y. H., and Ellenberger, T. (2005) Biochemistry 44,8312–8325

33. Scapin, G., Patel, S. B., Chung, C., Varnerin, J. P., Edmondson, S. D., Mas-

tracchio, A., Parmee, E. R., Singh, S. B., Becker, J. W., Van der Ploeg, L. H.,and Tota, M. R. (2004) Biochemistry 43, 6091–6100

34. Xu, R. X., Hassell, A. M., Vanderwall, D., Lambert, M. H., Holmes, W. D.,Luther, M. A., Rocque, W. J., Milburn, M. V., Zhao, Y., Ke, H., and Nolte,R. T. (2000) Science 288, 1822–1825

35. Xu, R. X., Rocque,W. J., Lambert, M. H., Vanderwall, D. E., Luther, M. A.,and Nolte, R. T. (2004) J. Mol. Biol. 337, 355–365

36. Lee, M. E., Markowitz, J., Lee, J. O., and Lee, H. (2002) FEBS Lett. 530,53–58

37. Huai, Q.,Wang, H., Sun, Y., Kim,H. Y., Liu, Y., andKe, H. (2003) Structure(Camb.) 11, 865–873

38. Huai, Q., Colicelli, J., and Ke, H. (2003) Biochemistry 42, 13220–1322639. Huai, Q., Wang, H., Zhang, W., Colman, R., Robinson, H., and Ke, H.

(2004) Proc. Natl. Acad. Sci. U. S. A. 101, 9624–962940. Fink, T. L., Francis, H., Beasley, A., Grimes, K. A., and Corbin, J. D. (1999)

J. Biol. Chem. 274, 24613–2462041. Li,W. K., Zhang, R. Y., and Xiao, P. G. (1996) Phytochemistry 43, 527–53042. Kuroda,M.,Mimaki, Y., Sashida, Y., Umegaki, E., Yamazaki,M., Chiba, K.,

Mohri, T., Kitahara, M., Yasuda, A., Naoi, N., Xu, Z. W., and Li, M. R.(2000) Planta Med. 66, 575–577

43. Xin, Z., Kim, E., Tian, Z., Lin, G., and Guo, Y. (2001) Chin. Sci. Bull. 46,1186–1190

44. Xin, Z., Kim, E. K., Lin, C. S., Liu, W. J., Tian, L., Yuan, Y. M., and Fu, J.(2003) Asian J. Androl. 5, 15–18

45. Zoraghi, R., Corbin, J. D., and Francis, S. H. (2006) J. Biol. Chem. 281,5553–5558

46. Turko, I. V., Ballard, S. A., Francis, S. H., and Corbin, J. D. (1999) Mol.Pharmacol. 56, 124–130

47. Nichols, M. R., and Morimoto, B. H. (2000) Mol. Pharmacol. 57,738–754

48. Ko,W., Shih, C., Lai, Y., Chen, J., andHuang, H. (2004) Biochem. Pharma-col. 68, 2087–2094

49. Barnes, S., Boersma, B., Patel, R., Kirk, P. M., Darley-Usmar, V. M., Kim,H., and Xu, J. (2000) Biofactors 12, 209–215

50. Fitzpatrick, L. A. (2003)Maturitas 44, S21–S2951. Limer, J., and Speirs, V. (2004) Breast Cancer Res. 6, 119–127

Structures of Phosphodiesterase-5

JULY 28, 2006 • VOLUME 281 • NUMBER 30 JOURNAL OF BIOLOGICAL CHEMISTRY 21479

by guest on March 19, 2016

http://ww

w.jbc.org/

Dow

nloaded from

Hengming KeFrancis, Jackie D. Corbin, Howard Robinson, Zhongcheng Xin, Guiting Lin and Huanchen Wang, Yudong Liu, Qing Huai, Jiwen Cai, Roya Zoraghi, Sharron H.

ENZYME FUNCTION AND DRUG DEVELOPMENTMultiple Conformations of Phosphodiesterase-5: IMPLICATIONS FOR

doi: 10.1074/jbc.M512527200 originally published online May 30, 20062006, 281:21469-21479.J. Biol. Chem. 

  10.1074/jbc.M512527200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/281/30/21469.full.html#ref-list-1

This article cites 43 references, 9 of which can be accessed free at

by guest on March 19, 2016

http://ww

w.jbc.org/

Dow

nloaded from